title: Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger.
abstract: Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2(S)-M2, and one module harbors the rtTA2(S)-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined.
authors.affiliation: Berlin University of Technology, Institute of Biotechnology, Department of Applied and Molecular Microbiology, Gustav-Meyer-Allee 25, 13355 Berlin, Germany. vera.meyer@tu-berlin.de
authors.forename: Vera
authors.initials: V
authors.lastname: Meyer
authors.forename: Franziska
authors.initials: F
authors.lastname: Wanka
authors.forename: Janneke
authors.initials: J
authors.lastname: van Gent
authors.forename: Mark
authors.initials: M
authors.lastname: Arentshorst
authors.forename: Cees A M J J
authors.initials: CA
authors.lastname: van den Hondel
authors.forename: Arthur F J
authors.initials: AF
authors.lastname: Ram
mesh_terms: D001234:Aspergillus niger / Q000235:genetics*; D002244:Carbon / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis*; D015966:Gene Expression Regulation, Fungal; D005827:Genetics, Microbial / Q000379:methods*; D016254:Mutagenesis, Insertional; D009584:Nitrogen / Q000378:metabolism; D011995:Recombination, Genetic; D015533:Transcriptional Activation
chemical_list: D005656:Fungal Proteins; D002244:Carbon; D009584:Nitrogen